Differentiation of Clostridium perfringens from Related Clostridia in Iron Milk Medium

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1 130 Journal of Food Protection, Vol. 48, No. 2, Pages (February 1985) Copyright International Association of Milk, Food, and Environmental Sanitarians Differentiation of Clostridium perfringens from Related Clostridia in Iron Milk Medium CARLOS ABEYTA, JR., 1 ANITA MICHALOVSKIS 2 and MARLEEN M. WEKELL 1 * U.S. Health and Human Services, Public Health Service, Food and Drug Administration, Seafood Products Research Center, Seattle, Wash and Institute for Food Science and Technology, College of Ocean and Fishery Sciences, University of Washington, Seattle, Washington (Received for publication June 4, 1984) ABSTRT The stormy fermentation reaction of Clostridium perfringens in iron milk medium was compared to that of several C. perfringenslike strains. These Clostridia, C. barati, C. perenne, C. absonum, and C. paraperfringens are very similar to C. perfringens on the basis of certain biochemical reactions and, consequently, are often difficult to distinguish from C. perfringens. Furthermore, these related Clostridia may also be present in foods. Results of this study demonstrate that after h of incubation at 45 C, only C. perfringens gave a positive reaction in iron milk with inocula as low as 22 cells/g. Some of the other strains began to show only gas production at h. After to 42 h some strains gave positive results and after 72 h all were positive. Enumeration of C. perfringens from food samples in iron milk medium by a 3tube most probable number (MPN) technique gave similar results to enumeration by plate count using ShahidiFerguson Perfringens (P) agar. Furthermore, a rapid positive response occurred after only 2 and 3 h incubation of iron milk inoculated with 10 8 and 10 7 cells/ml, respectively. The high selectivity, ease of identification and rapid growth of C. perfringens in iron milk make the iron milk MPN procedure a valuable assay for accurate enumeration and differentiation of C. perfringens from related Clostridia in food products. Identification of Clostridium perfringens is usually based on such conventional confirmation tests as nitrate reduction, motility, lactose fermentation, gelatin liquefaction, and gram stain (5). Other tests proposed to identify C. perfringens include acid phosphatase (12), reverse CAMP test (6), and the antitoxin halfplate method (14). Rapid screening procedures (4, 11), based on the stormy fermentation in iron milk have recently been developed for enumeration of C. perfringens. These methods (4, 11) rely solely on the rapid growth of C. perfringens at 45 C and on the stormy fermentation. Stormy fermentation in iron milk is highly indicative of the presence of C. per ' Food and Drug Administration. 2 University of Washington. fringens (1, 3, 4, 11) so that this reaction can be included as part of the presumptive confirmation procedure (5). St. John et al. (11) have suggested that C. perfringens could be enumerated in iron milk medium without other biochemical confirmation tests, for the combination of rapid growth in iron milk at 45 C and a stormy fermentation reaction was adequate for confirmation. The results of later studies support that view. Wekell et al. (13) and Abeyta (1), in studies of rat intestinal contents and fresh seafoods, respectively, found that all tubes demonstrating stormy fermentation within h of inoculation were confirmed as C. perfringens. In a study by Erickson et al. (4), using a similar medium with fortified litmus milk, C. perfringens could also be enumerated using the medium without confirmation. Some other clostridial strains occasionally isolated from foods have biochemical characteristics similar to C. perfringens. These similarities can sometimes result in falsepositive results (7). Several of these strains, C. paraperfringens, C. perenne, C. absonum, and C. barati, will grow rapidly at 45 C and can cause a stormy fermentation reaction in iron milk. This study was conducted to determine whether these C. perfringenslike strains could result in falsepositives when using the iron milk medium for enumeration of C. perfringens in foods. MATERIALS AND METHODS Cultures One strain of C. perfringens FD1 and four C. perfringenslike species (Clostridium paraperfringens, ATCC27639; Clostridium perenne, ATCC25782; Clostridium absonum ATCC27555; and Clostridium barati VPI44462) were obtained from S.M. Harmon (FDA, Center for Food Safety and Applied Nutrition, Washington, D.C ). Stock spore suspensions were stored until use in buffered trypticase peptone glucose yeast (TPY) broth with equal parts of glycerol salt solution at 72 C. Before testing, each culture was grown in TPY for h at 37 C. The rapidity of the stormy fermentation reaction depends on the initial population (in addition to strain type) (5); therefore, only vigorously growing cultures were used in this study. Seven other strains of C. perfringens (FD37, S45, FD29, FD21, S34, CDC 61 and FD2, obtained from S.M. Harmon) were treated as described and tested only for stormy fermentation.

2 IRON MILK MEDIUM TO DIFFERENTIATE CLOSTRIDIA 131 Media preparation The Trypticase Peptone lucose Yeast (TPY) broth was prepared by dissolving trypticase (50 g), peptone (5 g), yeast extract (20 g), glucose (4 g), disodium phosphate (5 g), and sodium thioglycollate (1 g) in 1 L of distilled water. The ph was adjusted to ph 7.3 with 1 N NaOH. The TPY broth was dispensed in 15ml portions into 20 x 150 mm screwcapped culture tubes and sterilized at 121 C for 8 min. The TPY broth has been found to support growth of C. perfringens better than cooked meat broth (Harmon, personal communication) and so was used as a growth medium for all Clostridia in this study. The iron milk medium (IMM, //) was prepared by placing 10 ml of fresh homogenized pasteurized milk and approximately 0.2 g of iron powder in each of several 16 x 150 mm screwcapped culture tubes. The medium was sterilized at 10 lb pressure at 116 C for 10 min and used within 2 h of preparation. ShahidiFerguson Perfringens (P) agar and Tryptic Soy Agar (TSA) were prepared according to label instructions (Difco) and used on the day of preparation. Enumeration of Clostridia in beef gravy All procedures were conducted using aseptic techniques. A 25g portion of canned beef gravy (FrancoAmerican, Campbell Soup Company) was transferred by pipet into an 800ml blender jar containing 225 ml of 0.1% peptone water and homogenized for 2 min at high speed using an Oster Homogenizer (Model #54841A). A 1.0ml portion of a hold culture (grown in TPY at 37 C) and diluted with 0.1% peptone water was added to the gravy homogenate and blended at high speed for 2 min at room temperature. For each clostridial strain, gravy samples were inoculated with cells ranging from approximately 1 x 10 3 to 1 x 10 4 cells per gram. Immediately the blended and inoculated gravy was diluted serially in 0.1% peptone solution. For enumeration using P agar, 0.1ml portions of serial dilutions were spread on duplicate plates (2). Plates were incubated at 37 C for 48 h. For enumeration using the iron milk MPN method 1.0ml portions of serial dilutions were added to each of 3 tubes per dilution as described (11). The iron milk tubes were incubated at 45 C for h. The hold culture used for inoculation was enumerated by plate counts using TSA agar. Duplicate pour plates containing 0.1 ml of serial decimal dilutions in 0.1% peptone water were incubated at 37 C. All P and TSA plates were incubated under anaerobic conditions using the as Pak System (Baltimore Biological Laboratory). The depth of the medium in the culture tubes and presence of iron powder provided sufficient anaerobiosis for growth of Clostridia in the iron milk medium. Metabolic reactions of Clostridia in iron milk medium All strains were grown in TPY at 37 C for h as described previously. After incubation, cultures grown in TPY broth were diluted serially (0.1% peptone) from approximately 1 x 10 7 to 1 x 10' cells per ml. Each dilution was enumerated using duplicate TSA pour plates incubated under anaerobic conditions. Oneml portions from each serial dilution were delivered into tubes of IMM. The IMM was incubated at 45 C, and inspected once per hour up to 10 h for signs of stormy fermentation. After 10 h, tubes of medium were inspected so that observations were made at,, and 72 h after inoculation. Biochemical confirmation tests Isolates from iron milk and P media were tested for motility, nitrate reduction, gelatin liquefaction, and lactose fermentation as outlined (2). RESULTS AND DISCUSSION C. perfringens growing on P agar is able to reduce sufite to sulfide and produce black colonies 1 to 2 mm in diameter. These black colonies are then surrounded by distinct white zones of precipitate 3 to 4 mm in diameter (10). The presence of the black colonies helps distinguish C. perfringens from other organisms such as Steptococcus faecalis, Proteus vulgaris, P. morganii, and Bacillus cereus that are not inhibited by the antibiotics in P agar, but only appear as pinpoint white colonies after incubation (11). Other Clostridia such as C. bifermentans, C. botulinum, C. parabotulinum, C. sporogenes, C. novyi, and C. haemolyticum will also show H 2 S and lecithinase production on P agar similar to C. perfringens. These strains, however, are motile and nonlactose fermenters and can be differentiated from C. perfringens by lactose fermentation and motility tests. It would not be possible to differentiate C. perfringens from the Clostridia used in this study by growth characteristics on P agar, lactose fermentation and motility. All Clostridia in this study were nonmotile, fermented nitrate to nitrite and gave the characteristic black colony surrounded by a zone of opaque precipitate on P agar. There were slight differences in the colony sizes as well as the diameter of the opaque precipitate zone surrounding the colonies. Most of the strains varied in colony size, being slightly smaller or larger than C. perfringens colonies. The opaque white zone surrounding C. absonum colonies was larger in diameter than those seen in the other strains. However, the differences in sizes of colonies and opaque white zones between these Clostridia grown on P agar would not be easily noticed by the untrained eye and would not be reliable for selectivity purposes. In this study, only C. perfringens and C. absonum were positive for gelatin liquefaction, but not all strains of C. absonum have the ability to liquefy gelatin in lactosegelatin medium. Since some heatresistant strains of C. perfringens can lose their ability to liquefy gelatin (9), the gelatin liquefaction test for the identification of C. perfringens and related strains is not always reliable. Stormy fermentation in iron milk results when there is an abundance of gas (C0 2 and H 2 ) that may be formed as a result of lactose fermentation. The gas fractures the acid clot into a spongy mass which rises above the medium surface. The acid clot is produced by the acidification of milk (lactic acid fermentation to a ph of 4.5) (8). Only C. perfringens exhibited a stormy fermentation in IMM before or at h of incubation at 45 C (Table 1). All 8 strains of C. perfringens used in this study were capable of producing this reaction within h. With the FD1 strain, stormy fermentation occurred as soon as 2 h of incubation with an inoculum of 1.7 x 10 s cells/ml, and at 9 h with a low inoculum level of 2.2 x 10 1 cells/ g. After h of incubation, C. barati showed a weak stormy fermentation, whereas C. perenne and C. paraperfringens produced only weak gas detected by the presence of small bubbles on the surface of the milk medium. After 42 h of incubation, C. paraperfringens, C. perenne, and C. absonum produced slight milk clotting. All strains after 72 h of incubation gave a stormy fermentation reaction (Table 2). For selective purposes, therefore, the iron milk must be evaluated for positive stormy fermentation for C. per JOURNAL OF FOOD PROTECTION. VOL. 48, FEBRUARY 1985

3 132 ABEYTA, MICHALOVSKIS AND WEKELL fringens before or at h of incubation at 45 C. Some of the C. perfringenslike strains in this study showed gas production only at h and a weak stormy fermentation after h of incubation. The weak stormy fermentation resembles the characteristic stormy fermentation of C. perfringens and may be misinterpreted as positive for C. perfringens if analyzed after h of incubation. Photographs taken (Fig. 13) of the typical stormy fermentation of C. perfringens in iron milk medium as well as production of gas, milk clotting, and weak stormy fermentation reactions of the other strains were helpful in visualizing the differences between the different reactions of C. perfringens and similar strains. The inoculum level of C. perfringens and related strains suspended into canned beef gravy ranged from 1.1 x 10 3 cells/ml to 1.1 x 10 4 cells/ml (Table 3). Plate counts of the different strains in P agar showed greater than 100% recoveries for C. perfringens, C. barati, C. absonum and an 83% recovery for C. perenne. Of all the strains tested, C. paraperfringens showed the poorest recovery, a 1000fold reduction. The cause for the decreased recoveries is not known, but could be responsible TABLE 1. Metabolic reactions of Clostridium perfringens FDI in IronMilk Medium. Level of inoculum" (cells per ml) Time (h) 1.7 xlo x xlo 6 1.6x10 s 1.6X " 2 d c "Inoculum level determined by plate counts on tryptic soy agar. b Acid clot formation,. c as formation,. d Stormy fermentation. TABLE 2. Strain Metabolic reactions of Clostridium perfringens and related strains in IronMilk Time Level of inoculum 13 (h) (cells per gram) 1.0 xlo 7 1.0X X XlO 3 Medium." 1.7X x 10' 9.4X X X10 C. perenne ATCC25782 c d e 1.3 xlo xlo xlo xlo x X 10 C. absonum ATCC27555 _f xlo 7 3.7x xl x xlo x 10' C. paraperfringens ATCC x X X X X X 10 C. barati VPI a At hrs strains of Clostridium perfringens (FD1, FD37, S45, FD29, FD21, S34, CDC 61 and FD2) were positive for stormy fermentation for all dilutions. b Inoculum level determined by plate counts on tryptic soy agar. c as formation, (). d Weak positive for stormy fermentation, (W). e Positive for stormy fermentation,. 'Negative for stormy fermentation,. JOURNAL OF FOOD PROTECTION. VOL. 48, FEBRUARY 1985

4 IRON MILK MEDIUM TO CLOSTRIDIA 133 Figure 1. Metabolic reactions of Clostridium perfringens compared to related Clostridia in Iron Milk Medium at hl45 C. Left tube, C. perfringens FD1, stormy fermentation; second tube from left, C. perenne ATCC25782, gas production, detected by the presence of small bubbles on the surface; third tube from left, C. absonum ATCC27555, no activity observed; forth tube from left, C. paraperfringens ATCC27639, gas production, detected by the presence of small bubbles on the surface. Figure 2. Metabolic reactions of Clostridium perfringens compared to related Clostridia in Iron Milk Medium at 42 hl45 C. Left tube, C. perfringens FD1, stormy fermentation; second tube from left, C. perenne ATCC25782, acid clot formation to stormy fermentation; third tube from left, C. paraperfringens ATCC27639, gas production, detected by the presence of small bubbles on the surface; fourth tube from left, C. absonum ATCC27555, acid clot formation. for substantial underestimates of numbers of these organisms. The most probable number for C. perfringens in iron milk medium after hr was 1.1 x 10 4 cells/ml, which was comparable to the P plate count of 5.6 x 10 4 cells/ml for C. perfringens. Since C. barati, C. perenne, C. absonum, and C. paraperfringens did not produce a stormy fermentation in IMM after 16 to h of incubation, the most probable numbers for these strains in iron milk medium could not be determined. CONCLUSIONS The iron milk medium method is more selective for differentiation of C. perfringens from similar strains such as C. barati, C. perenne, C. absonum, and C. paraperfringens than P agar. These C. perfringensmke strains would not give falsepositive results for C. perfringens in suspect foods by using iron milk medium incubated at 45 C for h. These other Clostridia, however, may give falsepositives on other media such as P because of their similarity in colony appearance and biochemical reactions. For enumeration purposes, the iron milk medium 3tube MPN method quantitates C. perfringens much more rapidly than would P agar. Results are obtained within h. In addition, iron milk medium can save money and time in medium preparation. Materials for iron milk medium are inexpensive, readily available, and decrease the need for inhibitors and confirmatory testing. Incubation of iron milk medium requires only a thermostatically controlled water bath held at 45 C, whereas P agar plates require antibiotics, anaerobic jars, gas generating kits, and anaerobic indicators, all which can add to the expense of the medium.

5 134 ABAYTA, MICHALOVSKIS AND WEKELL Further investigation of the use of iron milk medium is necessary for determining its suitability for support of growth of C. perfringens cells which may have been injured during food processing. Nevertheless, iron milk medium may be beneficial for routine procedures in quality control for the enumeration and differentiation of C. perfringens from competing organisms of different genera as well as from C. perfringens\iks strains. KNOWLEDMENT The authors gratefully acknowledge Linda C. Vernon and Patricia B. Potts for excellent secretarial support. REFERENCES Figure 3. Metabolic reactions of Clostridium perfringens compared to related Clostridia in Iron Milk Medium at 72 hl45 C. Left tube, C. perfringens FD1, stormy fermentation; second tube from left, C. perenne ATCC25782, stormy fermentation; third tube from left, C. absonum ATCC27555, acid, clot formation to stormy fermentation; fourth tube from left, C. paraperfringens ATCC27639, acid clot formation to stormy fermentation. TABLE 3. Quantitative analysis of Clostridium perfringens and related strains in beef gravy using iron milk media (IMM) and ShahadiFergusonperfringens (P) agar. Strain C. perfringens FD1 C. barati VPI44462 C. perenne ATCC25782 C. absonum ATCC27555 C. paraperfringens ATCC27639 Inocula" (cells/g) 1.1 XlO 4 1.1X XlO XlO X 10 3 P (cells/g) 5.6X XlO XlO 3 3.2X XlO 1 "Determined by plate counts on tryptic soy agar. b Negative for stormy fermentation at 16 h. IMM 3tube MPN at h (no./g) 1.1X10 4 _b 1. Abeyta, C, Jr Comparison of iron milk and official AO methods for enumeration of Clostridium perfringens from fresh seafoods. J. Assoc. Off. Anal. Chem. 66: 2. Association of Official Analytical Chemists Official Methods of Analysis, 13th ed. AO, Washington, D.C. 3. DeBoer, E., and E.M. Boot Comparison of methods for isolation and confirmation of Clostridium perfringens from spices and herbs. J. Food Prot. 46: Erickson, J.E., and R.H. Deibel New medium for rapid screening and enumeration of Clostridium perfringens in foods. Appl. Environ. Microbiol. 36: Food and Drug Administration Bacteriological analytical manual for foods. Association of Official Analytical Chemists, Washington, D.C. 6. Hansen, M.V., and L.P. Elliott New presumptive identification test for Clostridium perfringens: Reverse CAMP test. J. Clin. Microbiol. 12: Harmon, S.M., and D.A. Daulter Media for confirming Clostridium perfringens from food and feces. J. Food Prot. 41: MacFaddin, J.F Biochemical tests for identification of medical bacteria. Waverly Press, Inc., Baltimore, MD. pp Nishida, S., M. Seo, and M. Nakagawa Sporulation, heat resistance, and biological properties of Clostridium perfringens. Appl. Microbiol. 17: Shahidi, S.A., and A.R. Ferguson New qualitative and confirmatory media for the rapid analysis of food for Clostridium perfringens. Appl. Microbiol. 22: St. John. W.D., J.R. Matches, and M.M. Wekell Use of iron milk medium for enumeration of Clostridium perfringens. J. Assoc. Off. Anal. Chem. 65: Ueno, K., H. Fujii, T. Marui, J. Takahashi, T. Sugitani, T. Ushijima, and S. Suzuki Acid phosphatase in Clostridium perfringens. Japan. J. Microbiol. 14: Wekell, M.M., W.J. Hartman, and F.M. Dong Incidence of increased numbers of Clostridium perfringens in the intestinal tract of rats fed xylitol. J. Nutr. 110: Willis, A.T., and. Hobbs A medium for the identification of Clostridia producing opalescence in egg yolk emulsions. J. Pathol. Bacteriol. 75: